Chalcone isomerase

ABSTRACT

An isolated nucleic acid fragment encoding a chalcone isomerase is disclosed. Also of concern is the synthesis of a recombinant DNA construct encoding all or a portion of the chalcone isomerase, in sense or antisense orientation, wherein expression of the recombinant DNA construct results in production of altered levels of the chalcone isomerase in a transformed host cell.

[0001] This application claims the benefit of U.S. ProvisionalApplication No. 60/404,220, filed Aug. 16, 2002, the entire content ofwhich is herein incorporated by reference.

FIELD OF INVENTION

[0002] The field of invention relates to plant molecular biology, and inparticular, to nucleic acid fragments encoding chalcone isomerase inplants and seeds.

BACKGROUND OF INVENTION

[0003] All phenylpropanoids are derived from cinnamic acid, which isformed from phenylalanine by the action of phenylalanine ammonia-lyase,the branch point between primary and secondary metabolism. Isoflavonesrepresent a class of secondary metabolites produced mainly in legumes bythe phenylpropanoid biosynthesis pathway. The biosynthetic pathway forfree isoflavones and their relationship with several other classes ofphenylpropanoids is presented in FIG. 1.

[0004] Free isoflavones rarely accumulate to high levels in soybeans.Instead they are usually conjugated to carbohydrates or organic acids.Soybean seeds contain three types of isoflavones in four differentforms: the aglycones daidzein, genistein, and glycitein; the glucosidesdiadzin, genistin, and glycitin; the acetylglucosides6″-O-acetyldaidzin, 6″-O-acetylgenistin, and 6″-O-acetylglycitin; andthe malonylglucosides 6″-O-malonyidaidzin, 6″-O-malonylgenistin, and6″-O- malonylglycitin. It has been reported that the isoflavones foundin soybean seeds possess antihemolytic (Naim, M. et al., J. Agric. FoodChem. 24:1174-1177 (1976)), antifungal (Naim, M. et al., J. Agric. FoodChem. 22:806-810 (1974)), oestrogenic (Price, K.R. and Fenwick, G.R.Food Addit. Contam. 2:73-106 (1985)), tumor suppressing (Messina, M. andBarnes, S. J. Natl. Cancer lnst. 83:541-546 (1991); Peterson, G. et al.,Biochem. Biophys. Res. Commun. 179:661-667 (1991)), hypolipidemic(Mathur, K. et al., J. Nutr. 84:201-204 (1964)), and serum cholesterollowering (Sharma, R.D. Lipids 14:535-540 (1979)) effects. In addition,both epidemiological and dietary-intervention studies indicate that whenisoflavones in soybean seeds and in the subsequent protein products madefrom the seeds are part of the human dietary intake, these productsprovide many health benefits (Messina, M. J. Am. J. Clin. Nutr.70:439S-450S (1999)).

[0005] The content of isoflavones in soybean seeds, however, is quitevariable and is affected by both genetics and environmental conditionssuch as growing location and temperature during seed fill (Tsukamoto, C.et al., J. Agric. Food Chem. 43:1184-1192 (1995); Wang, H. and Murphy,P. A. J. Agric. Food Chem. 42:1674-1677 (1994)). In addition, isoflavonecontent in legumes can be stress-induced by pathogenic attack, wounding,high UV light exposure, and pollution (Dixon, R.A. and Paiva, N.L. ThePlant Cell 7:1085-1097 (1995)). To date, it has proven difficult todevelop soybean lines with consistently high levels of isoflavones.Moreover, lines reported to be low in isoflavone content produced normallevels of isoflavones when grown under standard cultural conditions(Kitamura, K. et al., Jap. J. Breed. 41:651-654 (1991)).

[0006] It is well known that many nuclear protein-encoding genes belongto gene families (evolutionary-related set of genes that encode similarproducts). Gene families in plants can be linked or dispersed throughoutthe genome, lying on separate chromosomes in unrelated locations. Thereare several probable reasons for their dispersion (i.e., allowspecialized expression and faster evolution). Soybeans are no exceptionand also contain gene families.

[0007] The isolation and cloning of genes associated with synthesis andmetabolism of isoflavones in soybean will afford the application ofmolecular techniques to achieve stable, high level accumulation ofisoflavones. In particular, chalcone isomerase (E.C. 5.5.1.6) whichcatalyzes the cyclization of chalcone (4,2′, 4′,6′-tetrahydroxychalcone) and 6′-deoxychalcone(4,2′,4′-trihydroxychalcone), both of which are synthesized by theupstream enzyme chalcone synthase, into 2S)-naringenin(5,7,4′-trihydroxyflavanone) and (2S)-5-deoxyflavanone(7,4′-dihydroxyflavanone), respectfully. Since both chalcone and6′-deoxychalcone spontaneously cyclize in solution to give enantiomericmixtures, chalcone isomerase guarantees formation of only thebiologically active (S)-isomer. A soybean chalcone isomerase haspreviously been identified (U.S. Pat. No. 6,054,636). The presentinvention provides novel chalcone isomerases from soybean (Glycine max).

SUMMARY OF INVENTION

[0008] The present invention includes isolated polynucleotidescomprising a nucleotide sequence encoding a polypeptide having chalconeisomerase activity wherein the amino acid sequence of the polypeptideand the amino acid sequence of SEQ ID NO:2, 4 or 6 have preferably atleast 80% sequence identity, preferably based on the Clustal method ofalignment. It is more preferred that the identity be at least 85%, evenmore preferred if the identity is at least 90%, and even more preferredthat the identity be at least 95%. The present invention also includesisolated polynucleotides comprising the full-length complement of thenucleotide sequence. More preferably, the present invention includesisolated polynucleotides encoding the polypeptide sequence of SEQ IDNO:2, 4 or 6 or nucleotide sequences comprising the nucleotide sequenceof SEQ ID NO:1, 3 or 5.

[0009] The present invention also includes:

[0010] in a preferred first embodiment, the present invention relates toan isolated polynucleotide comprising: (a) a nucleotide sequenceencoding a polypeptide having chalcone isomerase activity, wherein theamino acid sequence of the polypeptide and the amino acid sequence ofSEQ ID NO:2, 4 or 6 have at least 80%, 85%, 90%, or 95% identity,preferably based on the Clustal method of alignment, or (b) thecomplement of the nucleotide sequence of (a); the polypeptide preferablycomprises the amino acid sequence of SEQ ID NO:2, 4 or 6; the nucleotidesequence preferably comprises the nucleotide sequence of SEQ ID NO:1, 3or 5; the polypeptide preferably has chalcone isomerase activity;

[0011] in a preferred second embodiment, a vector comprising any of theisolated polynucleotides of the present invention;

[0012] in a preferred third embodiment, a recombinant DNA constructcomprising any of the isolated polynucleotides of the present inventionoperably linked to at least one regulatory sequence, and a cell, aplant, and a seed comprising the recombinant DNA construct;

[0013] in a preferred fourth embodiment, a method for transforming acell comprising transforming a cell with any of the isolatedpolynucleotides or recombinant DNA constructs of the present invention,and the cell transformed by this method; advantageously, the cell iseukaryotic, e.g., a yeast or plant cell, or prokaryotic, e.g., abacterium;

[0014] in a preferred fifth embodiment, a method for producing atransgenic plant comprising transforming a plant cell with any of theisolated polynucleotides or recombinant DNA constructs of the presentinvention and regenerating a plant from the transformed plant cell, atransgenic plant produced by this method, and seed obtained from thistransgenic plant;

[0015] in a preferred sixth embodiment, a first nucleotide sequencewhich contains at least 30 nucleotides, and wherein the first nucleotidesequence is comprised by another polynucleotide, wherein the otherpolynucleotide includes: (a) a second nucleotide sequence, wherein thesecond nucleotide sequence encodes a polypeptide having chalconeisomerase activity, wherein the amino acid sequence of the polypeptideand the amino acid sequence of SEQ ID NO:2, 4 or 6 have at least 80%sequence identity, or (b) the complement of the second nucleotidesequence of (a);

[0016] in a preferred seventh embodiment, an isolated polypeptidecomprising an amino acid sequence having chalcone isomerase activity,wherein the amino acid sequence and the amino acid sequence of SEQ IDNO:2, 4 or 6 have at least 80%, 85%, 90%, or 95% sequence identity,preferably based on the Clustal method of alignment; the amino acidsequence preferably comprises the amino acid sequence of SEQ ID NO:2, 4or 6; the polypeptide preferably has chalcone isomerase activity;

[0017] in a preferred eighth embodiment, a method for isolating apolypeptide comprising isolating the polypeptide from a cell or culturemedium of the cell, wherein the cell comprises a recombinant DNAconstruct comprising the polynucleotide of the present inventionoperably linked to at least one regulatory sequence.

BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCE LISTINGS

[0018] The invention can be more fully understood from the followingdetailed description and the accompanying drawings and Sequence Listingwhich form a part of this application.

[0019]FIG. 1 depicts the phenylpropanoid metabolic pathway illustratingthe biosynthesis of isoflavones.

[0020]FIG. 2 shows a comparison of: (i) the amino acid sequences of thechalcone isomerase encoded by the following: (a) nucleotide sequencederived from soybean clone src2c.pk001.k1 (SEQ ID NO:2), (b) nucleotidesequence derived from soybean clone sls1c.pk018.k17 (SEQ ID NO:4), (c)nucleotide sequence derived from soybean clone sah1c.pk002.f8 (SEQ IDNO:6); (ii) amino acid sequence of Phaseolus vulgaris chalcone isomerase(NCBI General Identifier No. 3041652; SEQ ID NO:7); and (iii) nucleotidesequence from Vitis vinifera chalcone isomerase (NCBI General IdentifierNo. 1705761; SEQ ID NO:8). Dashes are used by the program to maximizealignment of the sequences.

[0021] Table 1 lists the polypeptides that are described herein, thedesignation of the cDNA clones that comprise the nucleic acid fragmentsencoding polypeptides representing all or a substantial portion of thesepolypeptides, and the corresponding identifier (SEQ ID NO:) as used inthe attached Sequence Listing. The sequence descriptions and SequenceListing attached hereto comply with the rules governing nucleotideand/or amino acid sequence disclosures in patent applications as setforth in 37 C.F.R. §1.821-1.825. TABLE 1 Chalcone Isomerase SEQ ID NO:Amino Protein Clone Designation Nucleotide Acid Soybean polypeptidesimilar src2c.pk001.k1 1 2 to Phaseolus vulgaris chalcone isomeraseSoybean polypeptide similar sls1c.pk018.k17 3 4 to Phaseolus vulgarischalcone isomerase Soybean polypeptide similar sah1c.pk002.f8 5 6 toVitis vinifera chalcone isomerase

[0022] SEQ ID NO:7 is the amino acid sequence of a chalcone isomerasefrom Phaseolus vulgaris (NCBI General Identifier No. 3041652).

[0023] SEQ ID NO:8 is the amino acid sequence of a chalcone isomerasefrom Vitis vinifera (NCBI General Identifier No. 1705761).

[0024] The Sequence Listing contains the one letter code for nucleotidesequence characters and the three letter codes for amino acids asdefined in conformity with the IUPAC-IUBMB standards described inNucleic Acids Res. 13:3021-3030 (1985) and in the Biochemical J. 219(No. 2):345-373 (1984) which are herein incorporated by reference. Thesymbols and format used for nucleotide and amino acid sequence datacomply with the rules set forth in 37 C.F.R. §1.822.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[0025] The disclosure of each patent, patent application and non-patentliterature in the instant specification is incorporated herein byreference in its entirety.

[0026] In the context of this disclosure, a number of terms shall beutilized. “Chalcone isomerase” is also known as chalcone-flavanoneisomerase and CHI. These terms can be used interchangeably.

[0027] The terms “polynucleotide”, “polynucleotide sequence”, “nucleicacid sequence”, and “nucleic acid fragment”/“isolated nucleic acidfragment” are used interchangeably herein. These terms encompassnucleotide sequences and the like. A polynucleotide may be a polymer ofRNA or DNA that is single- or double- stranded, that optionally containssynthetic, non-natural or altered nucleotide bases. A polynucleotide inthe form of a polymer of DNA may be comprised of one or more segments ofcDNA, genomic DNA, synthetic DNA, or mixtures thereof. An isolatedpolynucleotide of the present invention may include preferably at least30 contiguous nucleotides, more preferably at least 40 contiguousnucleotides, most preferably at least 60 contiguous nucleotides derivedfrom SEQ ID NO:1, 3 or 5, or the complement of such sequences.

[0028] The term “isolated” refers to materials, such as nucleic acidmolecules and/or proteins, which are substantially free or otherwiseremoved from components that normally accompany or interact with thematerials in a naturally occurring environment. Isolated polynucleotidesmay be purified from a host cell in which they naturally occur.Conventional nucleic acid purification methods known to skilled artisansmay be used to obtain isolated polynucleotides. The term also embracesrecombinant polynucleotides and chemically synthesized polynucleotides.

[0029] The term “recombinant” means, for example, that a nucleic acidsequence is made by an artificial combination of two otherwise separatedsegments of sequence, e.g., by chemical synthesis or by the manipulationof isolated nucleic acids by genetic engineering techniques. A“recombinant DNA construct” comprises any of the isolatedpolynucleotides of the present invention operably linked to at least oneregulatory sequence.

[0030] As used herein, “contig” refers to a nucleotide sequence that isassembled from two or more constituent nucleotide sequences that sharecommon or overlapping regions of sequence homology. For example, thenucleotide sequences of two or more nucleic acid fragments can becompared and aligned in order to identify common or overlappingsequences. Where common or overlapping sequences exist between two ormore nucleic acid fragments, the sequences (and thus their correspondingnucleic acid fragments) can be assembled into a single contiguousnucleotide sequence.

[0031] As used herein, “substantially similar” refers to nucleic acidfragments wherein changes in one or more nucleotide bases results insubstitution of one or more amino acids, but do not affect thefunctional properties of the polypeptide encoded by the nucleotidesequence. “Substantially similar” also refers to nucleic acid fragmentswherein changes in one or more nucleotide bases does not affect theability of the nucleic acid fragment to mediate alteration of geneexpression by gene silencing through for example antisense orco-suppression technology. “Substantially similar” also refers tomodifications of the nucleic acid fragments of the instant inventionsuch as deletion or insertion of one or more nucleotides that do notsubstantially affect the functional properties of the resultingtranscript vis-a-vis the ability to mediate gene silencing or alterationof the functional properties of the resulting protein molecule. It istherefore understood that the invention encompasses more than thespecific exemplary nucleotide or amino acid sequences and includesfunctional equivalents thereof. The terms “substantially similar” and“corresponding substantially” are used interchangeably herein.

[0032] Substantially similar nucleic acid fragments may be selected byscreening nucleic acid fragments representing subfragments ormodifications of the nucleic acid fragments of the instant invention,wherein one or more nucleotides are substituted, deleted and/orinserted, for their ability to affect the level of the polypeptideencoded by the unmodified nucleic acid fragment in a plant or plantcell. For example, a substantially similar nucleic acid fragmentrepresenting at least 30 contiguous nucleotides, preferably at least 40contiguous nucleotides, most preferably at least 60 contiguousnucleotides derived from the instant nucleic acid fragment can beconstructed and introduced into a plant or plant cell. The level of thepolypeptide encoded by the unmodified nucleic acid fragment present in aplant or plant cell exposed to the substantially similar nucleicfragment can then be compared to the level of the polypeptide in a plantor plant cell that is not exposed to the substantially similar nucleicacid fragment.

[0033] For example, it is well known in the art that antisensesuppression and co-suppression of gene expression may be accomplishedusing nucleic acid fragments representing less than the entire codingregion of a gene, and by using nucleic acid fragments that do not share100% sequence identity with the gene to be suppressed. Moreover,alterations in a nucleic acid fragment which result in the production ofa chemically equivalent amino acid at a given site, but do not effectthe functional properties of the encoded polypeptide, are well known inthe art. Thus, a codon for the amino acid alanine, a hydrophobic aminoacid, may be substituted by a codon encoding another less hydrophobicresidue, such as glycine, or a more hydrophobic residue, such as valine,leucine, or isoleucine. Similarly, changes which result in substitutionof one negatively charged residue for another, such as aspartic acid forglutamic acid, or one positively charged residue for another, such aslysine for arginine, can also be expected to produce a functionallyequivalent product. Nucleotide changes which result in alteration of theN-terminal and C-terminal portions of the polypeptide molecule wouldalso not be expected to alter the activity of the polypeptide. Each ofthe proposed modifications is well within the routine skill in the art,as is determination of retention of biological activity of the encodedproducts. Consequently, an isolated polynucleotide comprising anucleotide sequence of at least 30 (preferably at least 40, mostpreferably at least 60) contiguous nucleotides derived from a nucleotidesequence of SEQ ID NO:1, 3 or 5, and the complement of such nucleotidesequences may be used to affect the expression and/or function of achalcone isomerase in a host cell. A method of using an isolatedpolynucleotide to affect the level of expression of a polypeptide in ahost cell (eukaryotic, such as plant or yeast, prokaryotic such asbacterial) may comprise the steps of: constructing an isolatedpolynucleotide of the present invention or an isolated recombinant DNAconstruct of the present invention; introducing the isolatedpolynucleotide or the isolated recombinant DNA construct into a hostcell; measuring the level of a polypeptide or enzyme activity in thehost cell containing the isolated polynucleotide; and comparing thelevel of a polypeptide or enzyme activity in the host cell containingthe isolated polynucleotide with the level of a polypeptide or enzymeactivity in a host cell that does not contain the isolatedpolynucleotide.

[0034] Moreover, substantially similar nucleic acid fragments may alsobe characterized by their ability to hybridize. Estimates of suchhomology are provided by either DNA-DNA or DNA-RNA hybridization underconditions of stringency as is well understood by those skilled in theart (Hames and Higgins, Eds. (1985) Nucleic Acid Hybridisation, IRLPress, Oxford, U.K.). Stringency conditions can be adjusted to screenfor moderately similar fragments, such as homologous sequences fromdistantly related organisms, to highly similar fragments, such as genesthat duplicate functional enzymes from closely related organisms.Post-hybridization washes determine stringency conditions. One set ofpreferred conditions uses a series of washes starting with 6×SSC, 0.5%SDS at room temperature for 15 min, then repeated with 2×SSC, 0.5% SDSat 45° C. for 30 min, and then repeated twice with 0.2×SSC, 0.5% SDS at50° C. for 30 min. A more preferred set of stringent conditions useshigher temperatures in which the washes are identical to those aboveexcept for the temperature of the final two 30 min washes in 0.2×SSC,0.5% SDS was increased to 60° C. Another preferred set of highlystringent conditions uses two final washes in 0.1×SSC, 0.1% SDS at 65°C.

[0035] Substantially similar nucleic acid fragments of the instantinvention may also be characterized by the percent identity of the aminoacid sequences that they encode to the amino acid sequences disclosedherein, as determined by algorithms commonly employed by those skilledin this art. Suitable nucleic acid fragments (isolated polynucleotidesof the present invention) encode polypeptides that are preferably atleast 70% identical, more preferably at least 80% identical to the aminoacid sequences reported herein. More preferred nucleic acid fragmentsencode amino acid sequences that are at least 85% identical to the aminoacid sequences reported herein. More preferred nucleic acid fragmentsencode amino acid sequences that are at least 90% identical to the aminoacid sequences reported herein. Most preferred are nucleic acidfragments that encode amino acid sequences that are at least 95%identical to the amino acid sequences reported herein. Suitable nucleicacid fragments not only have the above identities but typically encode apolypeptide having preferably at least 50 amino acids, more preferablyat least 100 amino acids, more preferably at least 150 amino acids,still more preferably at least 200 amino acids, and most preferably atleast 250 amino acids.

[0036] It is well understood by one skilled in the art that many levelsof sequence identity are useful in identifying related polypeptidesequences. Useful examples of percent identities are 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, or 95%, or any integer percentage from 50% to100%. Sequence alignments and percent identity calculations wereperformed using the Megalign program of the LASERGENE bioinformaticscomputing suite (DNASTAR Inc., Madison, Wis.). Multiple alignment of thesequences was performed using the Clustal method of alignment (Higginsand Sharp (1989) CABIOS. 5:151-153) with the default parameters (GAPPENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwisealignments using the Clustal method were KTUPLE 1, GAP PENALTY=3,WINDOW=5 and DIAGONALS SAVED=5.

[0037] A “substantial portion” of an amino acid or nucleotide sequencecomprises an amino acid or a nucleotide sequence that is sufficient toafford putative identification of the protein or gene that the aminoacid or nucleotide sequence comprises. Amino acid and nucleotidesequences can be evaluated either manually by one skilled in the art, orby using computer-based sequence comparison and identification toolsthat employ algorithms such as BLAST (Basic Local Alignment Search Tool;Altschul et al. (1993) J. Mol. Biol. 215:403-410; see also theexplanation of the BLAST alogarithm on the world wide web site for theNational Center for Biotechnology Information at the National Library ofMedicine of the National Institutes of Health). In general, a sequenceof ten or more contiguous amino acids or thirty or more contiguousnucleotides is necessary in order to putatively identify a polypeptideor nucleic acid sequence as homologous to a known protein or gene.Moreover, with respect to nucleotide sequences, gene-specificoligonucleotide probes comprising 30 or more contiguous nucleotides maybe used in sequence-dependent methods of gene identification (e.g.,Southern hybridization) and isolation (e.g., in situ hybridization ofbacterial colonies or bacteriophage plaques). In addition, shortoligonucleotides of 12 or more nucleotides may be used as amplificationprimers in PCR in order to obtain a particular nucleic acid fragmentcomprising the primers. Accordingly, a “substantial portion” of anucleotide sequence comprises a nucleotide sequence that will affordspecific identification and/or isolation of a nucleic acid fragmentcomprising the sequence. The instant specification teaches amino acidand nucleotide sequences encoding polypeptides that comprise one or moreparticular plant proteins. The skilled artisan, having the benefit ofthe sequences as reported herein, may now use all or a substantialportion of the disclosed sequences for purposes known to those skilledin this art. Accordingly, the instant invention includes the completesequences as reported in the accompanying Sequence Listing, as well assubstantial portions of those sequences as defined above.

[0038] “Codon degeneracy” refers to divergence in the genetic codepermitting variation of the nucleotide sequence without effecting theamino acid sequence of an encoded polypeptide. Accordingly, the instantinvention relates to any nucleic acid fragment comprising a nucleotidesequence that encodes all or a substantial portion of the amino acidsequences set forth herein. The skilled artisan is well aware of the“codon-bias” exhibited by a specific host cell in usage of nucleotidecodons to specify a given amino acid. Therefore, when synthesizing anucleic acid fragment for improved expression in a host cell, it isdesirable to design the nucleic acid fragment such that its frequency ofcodon usage approaches the frequency of preferred codon usage of thehost cell.

[0039] “Synthetic nucleic acid fragments” can be assembled fromoligonucleotide building blocks that are chemically synthesized usingprocedures known to those skilled in the art. These building blocks areligated and annealed to form larger nucleic acid fragments which maythen be enzymatically assembled to construct the entire desired nucleicacid fragment. “Chemically synthesized”, as related to a nucleic acidfragment, means that the component nucleotides were assembled in vitro.Manual chemical synthesis of nucleic acid fragments may be accomplishedusing well established procedures, or automated chemical synthesis canbe performed using one of a number of commercially available machines.Accordingly, the nucleic acid fragments can be tailored for optimal geneexpression based on optimization of the nucleotide sequence to reflectthe codon bias of the host cell. The skilled artisan appreciates thelikelihood of successful gene expression if codon usage is biasedtowards those codons favored by the host. Determination of preferredcodons can be based on a survey of genes derived from the host cellwhere sequence information is available.

[0040] “Gene” refers to a nucleic acid fragment that expresses aspecific protein, including regulatory sequences preceding (5′non-coding sequences) and following (3′ non-coding sequences) the codingsequence. “Native gene” refers to a gene as found in nature with its ownregulatory sequences. “Chimeric gene” refers any gene that is not anative gene, comprising regulatory and coding sequences that are notfound together in nature. Accordingly, a chimeric gene may compriseregulatory sequences and coding sequences that are derived fromdifferent sources, or regulatory sequences and coding sequences derivedfrom the same source, but arranged in a manner different than that foundin nature. “Endogenous gene” refers to a native gene in its naturallocation in the genome of an organism. A “foreign-gene” refers to a genenot normally found in the host organism, but that is introduced into thehost organism by gene transfer. Foreign genes can comprise native genesinserted into a non-native organism, recombinant DNA constructs, orchimeric genes. A “transgene” is a gene that has been introduced intothe genome by a transformation procedure.

[0041] “Coding sequence” refers to a nucleotide sequence that codes fora specific amino acid sequence. “Regulatory sequences” refer tonucleotide sequences located upstream (5′ non-coding sequences), within,or downstream (3′ non-coding sequences) of a coding sequence, and whichinfluence the transcription, RNA processing or stability, or translationof the associated coding sequence. Regulatory sequences may includepromoters, translation leader sequences, introns, and polyadenylationrecognition sequences.

[0042] “Promoter” refers to a nucleotide sequence capable of controllingthe expression of a coding sequence or functional RNA. In general, acoding sequence is located 3′ to a promoter sequence. The promotersequence consists of proximal and more distal upstream elements, thelatter elements often referred to as enhancers. Accordingly, an“enhancer” is a nucleotide sequence which can stimulate promoteractivity and may be an innate element of the promoter or a heterologouselement inserted to enhance the level or tissue-specificity of apromoter. Promoters may be derived in their entirety from a native gene,or may be composed of different elements derived from differentpromoters found in nature, or may even comprise synthetic nucleotidesegments. It is understood by those skilled in the art that differentpromoters may direct the expression of a gene in different tissues orcell types, or at different stages of development, or in response todifferent environmental conditions. Promoters which cause a nucleic acidfragment to be expressed in most cell types at most times are commonlyreferred to as “constitutive promoters”. New promoters of various typesuseful in plant cells are constantly being discovered; numerous examplesmay be found in the compilation by Okamuro and Goldberg (1989)Biochemistty of Plants 15:1-82. It is further recognized that since inmost cases the exact boundaries of regulatory sequences have not beencompletely defined, nucleic acid fragments of different lengths may haveidentical promoter activity.

[0043] “Translation leader sequence” refers to a nucleotide sequencelocated between the promoter sequence of a gene and the coding sequence.The translation leader sequence is present in the fully processed mRNAupstream of the translation start sequence. The translation leadersequence may affect processing of the primary transcript to mRNA, mRNAstability or translation efficiency. Examples of translation leadersequences have been described (Turner and Foster (1995) Mol. Biotechnol.3:225-236).

[0044] “3′ non-coding sequences” refer to nucleotide sequences locateddownstream of a coding sequence and include polyadenylation recognitionsequences and other sequences encoding regulatory signals capable ofaffecting mRNA processing or gene expression. The polyadenylation signalis usually characterized by affecting the addition of polyadenylic acidtracts to the 3′ end of the mRNA precursor. The use of different 3′non-coding sequences is exemplified by Ingelbrecht et al. (1989) PlantCell 1:671-680.

[0045] “RNA transcript” refers to the product resulting from RNApolymerase-catalyzed transcription of a DNA sequence. When the RNAtranscript is a perfect complementary copy of the DNA sequence, it isreferred to as the primary transcript or it may be a RNA sequencederived from posttranscriptional processing of the primary transcriptand is referred to as the mature RNA. “Messenger RNA (mRNA)” refers tothe RNA that is without introns and that can be translated intopolypeptides by the cell. “cDNA” refers to DNA that is complementary toand derived from an mRNA template. The cDNA can be single-stranded orconverted to double stranded form using, for example, the Klenowfragment of DNA polymerase 1. “Sense-RNA” refers to an RNA transcriptthat includes the mRNA and so can be translated into a polypeptide bythe cell. “Antisense RNA” refers to an RNA transcript that iscomplementary to all or part of a target primary transcript or mRNA andthat blocks the expression of a target gene (see U.S. Pat. No.5,107,065, incorporated herein by reference). The complementarity of anantisense RNA may be with any part of the specific nucleotide sequence,i.e., at the 5′ non-coding sequence, 3′ non-coding sequence, introns, orthe coding sequence. “Functional RNA” refers to sense RNA, antisenseRNA, ribozyme RNA, or other RNA that may not be translated but yet hasan effect on cellular processes.

[0046] The term “operably linked” refers to the association of two ormore nucleic acid fragments on a single polynucleotide so that thefunction of one is affected by the other. For example, a promoter isoperably linked with a coding sequence when it is capable of affectingthe expression of that coding sequence (i.e., that the coding sequenceis under the transcriptional control of the promoter). Coding sequencescan be operably linked to regulatory sequences in sense or antisenseorientation.

[0047] The term “expression”, as used herein, refers to thetranscription and stable accumulation of sense (mRNA) or antisense RNAderived from the nucleic acid fragment of the invention. Expression mayalso refer to translation of mRNA into a polypeptide. “Antisenseinhibition” refers to the production of antisense RNA transcriptscapable of suppressing the expression of the target protein.“Overexpression” refers to the production of a gene product intransgenic organisms that exceeds levels of production in normal ornon-transformed organisms. “Co-suppression” refers to the production ofsense RNA transcripts capable of suppressing the expression of identicalor substantially similar foreign or endogenous genes (U.S. Pat. No.5,231,020, incorporated herein by reference).

[0048] A “protein” or “polypeptide” is a chain of amino acids arrangedin a specific order determined by the coding sequence in apolynucleotide encoding the polypeptide. Each protein or polypeptide hasa unique function.

[0049] “Altered levels” or “altered expression” refers to the productionof gene product(s) in transgenic organisms in amounts or proportionsthat differ from that of normal or non-transformed organisms.

[0050] “Mature protein” or the term “mature” when used in describing aprotein refers to a post-translationally processed polypeptide; i.e.,one from which any pre- or propeptides present in the primarytranslation product have been removed. “Precursor protein” or the term“precursor” when used in describing a protein refers to the primaryproduct of translation of mRNA; i.e., with pre- and propeptides stillpresent. Pre- and propeptides may be but are not limited tointracellular localization signals.

[0051] A “chloroplast transit peptide” is an amino acid sequence whichis translated in conjunction with a protein and directs the protein tothe chloroplast or other plastid types present in the cell in which theprotein is made. “Chloroplast transit sequence” refers to a nucleotidesequence that encodes a chloroplast transit peptide. A “signal peptide”is an amino acid sequence which is translated in conjunction with aprotein and directs the protein to the secretory system (Chrispeels(1991) Ann. Rev. Plant Phys. Plant Mol. Biol. 42:21-53). If the proteinis to be directed to a vacuole, a vacuolar targeting signal (supra) canfurther be added, or if to the endoplasmic reticulum, an endoplasmicreticulum retention signal (supra) may be added. If the protein is to bedirected to the nucleus, any signal peptide present should be removedand instead a nuclear localization signal included (Raikhel (1992)

[0052] Plant Phys. 100:1627-1632).

[0053] “Transformation” refers to the transfer of a nucleic acidfragment into the genome of a host organism. Host organisms containingthe transformed nucleic acid fragments are referred to as “transgenic”organisms. Examples of methods of plant transformation includeAgrobacterium-mediated transformation (De Blaere et al. (1987) Meth.Enzymol. 143:277; Ishida Y. et al. (1996) Nature Biotech. 14:745-750)and particle-accelerated or “gene gun” transformation technology (Kleinet al. (1987) Nature (London) 327:70-73; U.S. Pat. No. 4,945,050,incorporated herein by reference). Thus, isolated polynucleotides of thepresent invention can be incorporated into recombinant constructs,typically DNA constructs, capable of introduction into and replicationin a host cell. Such a construct can be a vector that includes areplication system and sequences that are capable of transcription andtranslation of a polypeptide-encoding sequence in a given host cell. Anumber of vectors suitable for stable transfection of plant cells or forthe establishment of transgenic plants have been described in, e.g.,Pouwels et al., Cloning Vectors: A Laboratory Manual, 1985, supp. 1987;Weissbach and Weissbach, Methods for Plant Molecular Biology, AcademicPress, 1989; and Flevin et al., Plant Molecular Biology Manual, KluwerAcademic Publishers, 1990. Typically, plant expression vectors include,for example, one or more cloned plant genes under the transcriptionalcontrol of 5′ and 3′ regulatory sequences and a dominant selectablemarker. Such plant expression vectors also can contain a promoterregulatory region (e.g., a regulatory region controlling inducible orconstitutive, environmentally- or developmentally-regulated, or cell- ortissue-specific expression), a transcription initiation start site, aribosome binding site, an RNA processing signal, a transcriptiontermination site, and/or a polyadenylation signal.

[0054] “Stable transformation” refers to the transfer of a nucleic acidfragment into a genome of a host organism, including both nuclear andorganellar genomes, resulting in genetically stable inheritance. Incontrast, “transient transformation” refers to the transfer of a nucleicacid fragment into the nucleus, or DNA-containing organelle, of a hostorganism resulting in gene expression without integration or stableinheritance. Host organisms containing the transformed nucleic acidfragments are referred to as “transgenic” organisms. The term“transformation” as used herein refers to both stable transformation andtransient transformation.

[0055] The terms “recombinant construct”, “expression construct” and“recombinant expression construct” are used interchangeably herein.These terms refer to a functional unit of genetic material that can beinserted into the genome of a cell using standard methodology well knownto one skilled in the art. Such construct may be used by itself or maybe used in conjunction with a vector. If a vector is used, the choice ofvector is dependent upon the method that will be used to transform hostplants as is well known to those skilled in the art.

[0056] Standard recombinant DNA and molecular cloning techniques usedherein are well known in the art and are described more fully inSambrook et al. Molecular Cloning: A Laboratory Manual; Cold SpringHarbor Laboratory Press: Cold Spring Harbor, 1989 (hereinafter“Maniatis”).

[0057] “Motifs” or “subsequences” refer to short regions of conservedsequences of nucleic acids or amino acids that comprise part of a longersequence. For example, it is expected that such conserved subsequenceswould be important for function, and could be used to identify newhomologues in plants. It is expected that some or all of the elementsmay be found in a homologue. Also, it is expected that one or two of theconserved amino acids in any given motif may differ in a true homologue.

[0058] “PCR” or “polymerase chain reaction” is well known by thoseskilled in the art as a technique used for the amplification of specificDNA segments (U.S. Pat. Nos. 4,683,195 and 4,800,159).

[0059] It is believed that the polynucleotides of the instant inventionmay be used to create transgenic plants where the chalcone isomeraselevels are altered with respect to non-transgenic plants which wouldresult in plants with an altered phenotype.

[0060] Furthermore, overexpression of chalcone isomerase may result inan increase of compounds in flavonoid classes. More specifically,chalcone isomerase overexpression may result in an increase in levels of(2S)-naringenin (5,7,4′- trihydroxyflavanone) and (2S)-5-deoxyflavanone(7,4′-dihydroxyflavanone), precursors in the biosynthetic pathwaysleading to isoflavone, flavone and dihydroflavanol (which uponcontinuation leads to anthocyanin and flavanols) synthesis (see FIG. 1).Increased isoflavone content in legumes has been shown to be associatedwith beneficial health effects in humans.

[0061] The present invention includes an isolated polynucleotidecomprising a nucleotide sequence encoding a chalcone isomerasepolypeptide having at least 80% identity, based on the Clustal method ofalignment, when compared to a polypeptide of SEQ ID NO:2, 4 or 6.

[0062] This invention also includes to the isolated complement of suchpolynucleotides, wherein preferably the complement and thepolynucleotide consist of the same number of nucleotides, and thenucleotide sequences of the complement and the polynucleotide have 100%complementarity.

[0063] Nucleic acid fragments encoding at least a portion of severalchalcone isomerase proteins have been isolated and identified bycomparison of random plant cDNA sequences to public databases containingnucleotide and protein sequences using the BLAST algorithms well knownto those skilled in the art. The nucleic acid fragments of the instantinvention may be used to isolate cDNAs and genes encoding homologousproteins from the same or other plant species. Isolation of homologousgenes using sequence-dependent protocols is well known in the art.Examples of sequence-dependent protocols include, but are not limitedto, methods of nucleic acid hybridization, and methods of DNA and RNAamplification as exemplified by various uses of nucleic acidamplification technologies (e.g., polymerase chain reaction, ligasechain reaction).

[0064] For example, genes encoding other chalcone isomerase, either ascDNAs or genomic DNAs, could be isolated directly by using all or aportion of the instant nucleic acid fragments as DNA hybridizationprobes to screen libraries from any desired plant employing methodologywell known to those skilled in the art. Specific oligonucleotide probesbased upon the instant nucleic acid sequences can be designed andsynthesized by methods known in the art (Maniatis). Moreover, an entiresequence can be used directly to synthesize DNA probes by methods knownto the skilled artisan such as random primer DNA labeling, nicktranslation, end-labeling techniques, or RNA probes using available invitro transcription systems. In addition, specific primers can bedesigned and used to amplify a part or all of the instant sequences. Theresulting amplification products can be labeled directly duringamplification reactions or labeled after amplification reactions, andused as probes to isolate full length cDNA or genomic fragments underconditions of appropriate stringency.

[0065] In addition, two short segments of the instant nucleic acidfragments may be used in polymerase chain reaction protocols to amplifylonger nucleic acid fragments encoding homologous genes from DNA or RNA.The polymerase chain reaction may also be performed on a library ofcloned nucleic acid fragments wherein the sequence of one primer isderived from the instant nucleic acid fragments, and the sequence of theother primer takes advantage of the presence of the polyadenylic acidtracts to the 3′ end of the mRNA precursor encoding plant genes.Alternatively, the second primer sequence may be based upon sequencesderived from the cloning vector. For example, the skilled artisan canfollow the RACE protocol (Frohman et al. (1988) Proc. Natl. Acad. Sci.USA 85:8998-9002) to generate cDNAs by using PCR to amplify copies ofthe region between a single point in the transcript and the 3′ or 5′end. Primers oriented in the 3′ and 5′ directions can be designed fromthe instant sequences. Using commercially available 3′ RACE or 5′ RACEsystems (BRL), specific 3′ or 5′ cDNA fragments can be isolated (Oharaet al. (1989) Proc. Natl. Acad. Sci. USA 86:5673-5677; Loh et al. (1989)Science 243:217-220). Products generated by the 3′ and 5′ RACEprocedures can be combined to generate full-length cDNAs (Frohman andMartin (1989) Techniques 1:165). Consequently, a polynucleotidecomprising a nucleotide sequence of preferably at least 30 (morepreferably at least 40, most preferably at least 60) contiguousnucleotides derived from a nucleotide sequence of SEQ ID NOs:1, 3 or 5and the complement of such nucleotide sequences may be used in suchmethods to obtain a nucleic acid fragment encoding a substantial portionof an amino acid sequence of a polypeptide.

[0066] Availability of the instant nucleotide and deduced amino acidsequences facilitates immunological screening of cDNA expressionlibraries. Synthetic peptides representing portions of the instant aminoacid sequences may be synthesized. These peptides can be used toimmunize animals to produce polyclonal or monoclonal antibodies withspecificity for peptides or proteins comprising the amino acidsequences. These antibodies can be then be used to screen cDNAexpression libraries to isolate full-length cDNA clones of interest(Lerner (1984) Adv. Immunol. 36:1-34; Maniatis).

[0067] In another preferred embodiment, this invention includes virusesand host cells comprising either the recombinant DNA constructs of theinvention as described herein or isolated polynucleotides of theinvention as described herein. Examples of host cells which can be usedto practice the invention include, but are not limited to, yeast,bacteria, and plants.

[0068] The regeneration, development and cultivation of plants fromsingle plant protoplast transformants or from various transformedexplants is well known in the art (Weissbach and Weissbach, In, Methodsfor Plant Molecular Biology, (Eds.), Academic Press, Inc., San Diego,Calif. (1988)). This regeneration and growth process typically includesthe steps of selection of transformed cells, culturing thoseindividualized cells through the usual stages of embryonic developmentthrough the rooted plantlet stage. Transgenic embryos and seeds aresimilarly regenerated. The resulting transgenic rooted shoots arethereafter planted in an appropriate plant growth medium such as soil.

[0069] The development or regeneration of plants containing the foreign,exogenous gene that encodes a protein of interest is well known in theart. Preferably, the regenerated plants are self-pollinated to providehomozygous transgenic plants. Otherwise, pollen obtained from theregenerated plants is crossed to seed-grown plants of agronomicallyimportant lines. Conversely, pollen from plants of these important linesis used to pollinate regenerated plants. A transgenic plant of thepresent invention containing a desired polypeptide is cultivated usingmethods well known to one skilled in the art.

[0070] There are a variety of methods for the regeneration of plantsfrom plant tissue. The particular method of regeneration will depend onthe starting plant tissue and the particular plant species to beregenerated. Methods for transforming dicots, primarily by use ofAgrobacterium tumefaciens, and obtaining transgenic plants have beenpublished for cotton (U.S. Pat. No. 5,004,863, U.S. Pat. No. 5,159,135,U.S. Pat. No. 5,518,908); soybean (U.S. Pat. No. 5,569,834, U.S. Pat.No. 5,416,011, McCabe et. al., Bio/Technology6:923 (1988), Christou etal., Plant Physiol. 87:671-674 (1988)); Brassica (U.S. Pat. No.5,463,174); peanut (Cheng et al., Plant Cell Rep. 15:653-657 (1996),McKently et al., Plant Cell Rep. 14:699-703 (1995)); papaya; and pea(Grant et al., Plant Cell Rep. 15:254-258, (1995)).

[0071] Transformation of monocotyledons using electroporation, particlebombardment, and Agrobacterium have also been reported. Transformationand plant regeneration have been achieved in asparagus (Bytebier et al.,Proc. Natl. Acad. Sci. (USA) 84:5354, (1987)); barley (Wan and Lemaux,Plant Physiol 104:37 (1994)); Zea mays (Rhodes et al., Science 240:204(1988), Gordon-Kamm et al., Plant Cell 2:603-618 (1990), Fromm et al.,Bio/Technology 8:833 (1990), Koziel et al., Bio/Technology 11: 194,(1993), Armstrong et al., Crop Science 35:550-557 (1995)); oat (Somerset al., Bio/Technology 10: 15 89 (1992)); orchard grass (Horn et al.,Plant Cell Rep. 7:469 (1988)); rice (Toriyama et al., TheorAppL Genet.205:34, (1986); Part et al., Plant Mol. Biol. 32:1135-1148, (1996);Abedinia et al., Aust. J. Plant Physiol. 24:133-141 (1997); Zhang andWu, Theor. Appl. Genet. 76:835 (1988); Zhang et al. Plant Cell Rep.7:379, (1988); Battraw and Hall, Plant Sci. 86:191-202 (1992); Christouet al., Bio/Technology 9:957 (1991)); rye (De la Pena et al., Nature325:274 (1987)); sugarcane (Bower and Birch, Plant J. 2:409 (1992));tall fescue (Wang et al., Bio/Technology 10:691 (1992)), and wheat(Vasil et al., Bio/Technology 10:667 (1992); U.S. Patent No. 5,631,152).

[0072] Assays for gene expression based on the transient expression ofcloned nucleic acid constructs have been developed by introducing thenucleic acid molecules into plant cells by polyethylene glycoltreatment, electroporation, or particle bombardment (Marcotte et al.,Nature 335:454-457 (1988); Marcotte et al., Plant Cell 1:523-532 (1989);McCarty et al., Cell 66:895-905 (1991); Hattori et al., Genes Dev.6:609-618 (1992); Goff et al., EMBO J. 9:2517-2522 (1990)).

[0073] Transient expression systems may be used to functionally dissectgene constructs (see generally, Maliga et al., Methods in PlantMolecular Biology, Cold Spring Harbor Press (1995)). It is understoodthat any of the nucleic acid molecules of the present invention can beintroduced into a plant cell in a permanent or transient manner incombination with other genetic elements such as vectors, promoters,enhancers etc.

[0074] In addition to the above discussed procedures, practitioners arefamiliar with the standard resource materials which describe specificconditions and procedures for the construction, manipulation andisolation of macromolecules (e.g., DNA molecules, plasmids, etc.),generation of recombinant organisms and the screening and isolating ofclones, (see for example, Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Press (1989); Maliga et al.,Methods in Plant Molecular Biology, Cold Spring Harbor Press (1995);Birren et al., Genome Analysis: Detecting Genes, 1, Cold Spring Harbor,New York (1998); Birren et al., Genome Analysis: Analyzing DNA, 2, ColdSpring Harbor, New York (1998); Plant Molecular Biology: A LaboratoryManual, eds. Clark, Springer, New York (1997)).

[0075] Overexpression of the proteins of the instant invention may beaccomplished by first constructing a recombinant DNA construct in whichthe coding region is operably linked to a promoter capable of directingexpression of a gene in the desired tissues at the desired stage ofdevelopment. The recombinant DNA construct may comprise promotersequences and translation leader sequences derived from the same genes.3′ Non-coding sequences encoding transcription termination signals mayalso be provided. The instant recombinant DNA construct may alsocomprise one or more introns in order to facilitate gene expression.

[0076] Plasmid vectors comprising the instant isolated polynucleotide(s)(or recombinant DNA construct(s)) may be constructed. The choice ofplasmid vector is dependent upon the method that will be used totransform host plants. The skilled artisan is well aware of the geneticelements that must be present on the plasmid vector in order tosuccessfully transform, select and propagate host cells containing therecombinant DNA construct or chimeric gene. The skilled artisan willalso recognize that different independent transformation events willresult in different levels and patterns of expression (Jones et al.(1985) EMBO J. 4:2411-2418; De Almeida et al. (1989) Mol. Gen. Genetics218:78-86), and thus that multiple events must be screened in order toobtain lines displaying the desired expression level and pattern. Suchscreening may be accomplished by Southern analysis of DNA, Northernanalysis of mRNA expression, Western analysis of protein expression, orphenotypic analysis.

[0077] For some applications it may be useful to direct the instantpolypeptides to different cellular compartments, or to facilitate itssecretion from the cell. It is thus envisioned that the recombinant DNAconstruct(s) described above may be further supplemented by directingthe coding sequence to encode the instant polypeptides with appropriateintracellular targeting sequences such as transit sequences (Keegstra(1989) Cell 56:247-253), signal sequences or sequences encodingendoplasmic reticulum localization (Chrispeels (1991) Ann. Rev. PlantPhys. Plant Mol. Biol. 42:21-53), or nuclear localization signals(Raikhel (1992) Plant Phys. 100:1627-1632) with or without removingtargeting sequences that are already present. While the references citedgive examples of each of these, the list is not exhaustive and moretargeting signals of use may be discovered in the future.

[0078] It may also be desirable to reduce or eliminate expression ofgenes encoding the instant polypeptides in plants for some applications.In order to accomplish this, a recombinant DNA construct designed forco-suppression of the instant polypeptide can be constructed by linkinga gene or gene fragment encoding that polypeptide to plant promotersequences. Alternatively, a recombinant DNA construct designed toexpress antisense RNA for all or part of the instant nucleic acidfragment can be constructed by linking the gene or gene fragment inreverse orientation to plant promoter sequences. Either theco-suppression or antisense recombinant DNA constructs could beintroduced into plants via transformation wherein expression of thecorresponding endogenous genes are reduced or eliminated.

[0079] Molecular genetic solutions to the generation of plants withaltered gene expression have a decided advantage over more traditionalplant breeding approaches. Changes in plant phenotypes can be producedby specifically inhibiting expression of one or more genes by antisenseinhibition or cosuppression (U.S. Pat. Nos. 5,190,931, 5,107,065 and5,283,323). An antisense or cosuppression construct would act as adominant negative regulator of gene activity. While conventionalmutations can yield negative regulation of gene activity these effectsare most likely recessive. The dominant negative regulation availablewith a transgenic approach may be advantageous from a breedingperspective. In addition, the ability to restrict the expression of aspecific phenotype to the reproductive tissues of the plant by the useof tissue specific promoters may confer agronomic advantages relative toconventional mutations which may have an effect in all tissues in whicha mutant gene is ordinarily expressed.

[0080] The person skilled in the art will know that specialconsiderations are associated with the use of antisense or cosuppressiontechnologies in order to reduce expression of particular genes. Forexample, the proper level of expression of sense or antisense genes mayrequire the use of different recombinant DNA constructs utilizingdifferent regulatory elements known to the skilled artisan. Oncetransgenic plants are obtained by one of the methods described above, itwill be necessary to screen individual transgenics for those that mosteffectively display the desired phenotype. Accordingly, the skilledartisan will develop methods for screening large numbers oftransformants. The nature of these screens will generally be chosen onpractical grounds. For example, one can screen by looking for changes ingene expression by using antibodies specific for the protein encoded bythe gene being suppressed, or one could establish assays thatspecifically measure enzyme activity. A preferred method will be onewhich allows large numbers of samples to be processed rapidly, since itwill be expected that a large number of transformants will be negativefor the desired phenotype.

[0081] In another preferred embodiment, the present invention includes achalcone isomerase polypeptide having an amino acid sequence that is atleast 80% identical, based on the Clustal method of alignment, to apolypeptide of SEQ ID NO:2, 4 or 6.

[0082] The instant polypeptides (or portions thereof) may be produced inheterologous host cells, particularly in the cells of microbial hosts,and can be used to prepare antibodies to these proteins by methods wellknown to those skilled in the art. The antibodies are useful fordetecting the polypeptides of the instant invention in situ in cells orin vitro in cell extracts. Preferred heterologous host cells forproduction of the instant polypeptides are microbial hosts. Microbialexpression systems and expression vectors containing regulatorysequences that direct high level expression of foreign proteins are wellknown to those skilled in the art. Any of these could be used toconstruct a recombinant DNA construct for production of the instantpolypeptides. This recombinant DNA construct could then be introducedinto appropriate microorganisms via transformation to provide high levelexpression of the encoded chalcone isomerase. An example of a vector forhigh level expression of the instant polypeptides in a bacterial host isprovided (Example 6).

[0083] All or a substantial portion of the polynucleotides of theinstant invention may also be used as probes for genetically andphysically mapping the genes that they are a part of, and used asmarkers for traits linked to those genes. Such information may be usefulin plant breeding in order to develop lines with desired phenotypes. Forexample, the instant nucleic acid fragments may be used as restrictionfragment length polymorphism (RFLP) markers. Southern blots (Maniatis)of restriction-digested plant genomic DNA may be probed with the nucleicacid fragments of the instant invention. The resulting banding patternsmay then be subjected to genetic analyses using computer programs suchas MapMaker (Lander et al. (1987) Genomics 1:174-181) in order toconstruct a genetic map. In addition, the nucleic acid fragments of theinstant invention may be used to probe Southern blots containingrestriction endonuclease-treated genomic DNAs of a set of individualsrepresenting parent and progeny of a defined genetic cross. Segregationof the DNA polymorphisms is noted and used to calculate the position ofthe instant nucleic acid sequence in the genetic map previously obtainedusing this population (Botstein et al. (1980) Am. J. Hum. Genet32:314-331).

[0084] The production and use of plant gene-derived probes for use ingenetic mapping is described in Bernatzky and Tanksley (1986) Plant Mol.Biol. Reporter 4:37-41. Numerous publications describe genetic mappingof specific cDNA clones using the methodology outlined above orvariations thereof. For example, F2 intercross populations, backcrosspopulations, randomly mated populations, near isogenic lines, and othersets of individuals may be used for mapping. Such methodologies are wellknown to those skilled in the art.

[0085] Nucleic acid probes derived from the instant nucleic acidsequences may also be used for physical mapping (i.e., placement ofsequences on physical maps; see Hoheisel et al. In: Nonmammalian GenomicAnalysis: A Practical Guide, Academic press 1996, pp. 319-346, andreferences cited therein).

[0086] Nucleic acid probes derived from the instant nucleic acidsequences may be used in direct fluorescence in situ hybridization(FISH) mapping (Trask (1991) Trends Genet 7:149-154). Although currentmethods of FISH mapping favor use of large clones (several kb to severalhundred kb; see Laan et al. (1995) Genome Res. 5:13-20), improvements insensitivity may allow performance of FISH mapping using shorter probes.

[0087] A variety of nucleic acid amplification-based methods of geneticand physical mapping may be carried out using the instant nucleic acidsequences. Examples include allele-specific amplification (Kazazian(1989) J. Lab. Clin. Med. 11:95-96), polymorphism of PCR-amplifiedfragments (CAPS; Sheffield et al. (1993) Genomics 16:325-332),allele-specific ligation (Landegren et al. (1988) Science241:1077-1080), nucleotide extension reactions (Sokolov (1990) NucleicAcid Res. 18:3671), Radiation Hybrid Mapping (Walter et al. (1997) NatGenet 7:22-28) and Happy Mapping (Dear and Cook (1989) Nucleic Acid Res.17:6795-6807). For these methods, the sequence of a nucleic acidfragment is used to design and produce primer pairs for use in theamplification reaction or in primer extension reactions. The design ofsuch primers is well known to those skilled in the art. In methodsemploying PCR-based genetic mapping, it may be necessary to identify DNAsequence differences between the parents of the mapping cross in theregion corresponding to the instant nucleic acid sequence. This,however, is generally not necessary for mapping methods.

[0088] Loss of function mutant phenotypes may be identified for theinstant cDNA clones either by targeted gene disruption protocols or byidentifying specific mutants for these genes contained in a maizepopulation carrying mutations in all possible genes (Ballinger andBenzer (1989) Proc. Natl. Acad. Sci USA 86:9402-9406; Koes et al. (1995)Proc. Natl. Acad. Sci USA 92:8149-8153; Bensen et al. (1995) Plant Cell7:75-84). The latter approach may be accomplished in two ways. First,short segments of the instant nucleic acid fragments may be used inpolymerase chain reaction protocols in conjunction with a mutation tagsequence primer on DNAs prepared from a population of plants in whichMutator transposons or some other mutation-causing DNA element has beenintroduced (see Bensen, supra). The amplification of a specific DNAfragment with these primers indicates the insertion of the mutation tagelement in or near the plant gene encoding the instant polypeptides.Alternatively, the instant nucleic acid fragment may be used as ahybridization probe against PCR amplification products generated fromthe mutation population using the mutation tag sequence primer inconjunction with an arbitrary genomic site primer, such as that for arestriction enzyme site-anchored synthetic adaptor. With either method,a plant containing a mutation in the endogenous gene encoding theinstant polypeptides can be identified and obtained. This mutant plantcan then be used to determine or confirm the natural function of theinstant polypeptides disclosed herein.

EXAMPLES

[0089] The present invention is further illustrated in the followingExamples, in which parts and percentages are by weight and degrees areCelsius, unless otherwise stated. It should be understood that theseExamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only. From the above discussion and theseExamples, one skilled in the art can ascertain the essentialcharacteristics of this invention, and without departing from the spiritand scope thereof, can make various changes and modifications of theinvention to adapt it to various usages and conditions. Thus, variousmodifications of the invention in addition to those shown and describedherein will be apparent to those skilled in the art from the foregoingdescription. Such modifications are also intended to fall within thescope of the appended claims.

Example 1 Composition of cDNA Libraries; Isolation and Sequencing ofcDNA Clones

[0090] cDNA libraries representing mRNAs from various soybean (Glycinemax L.) tissues were prepared. The characteristics of the libraries aredescribed below. TABLE 2 cDNA Libraries from Soybean Library TissueClone src2c Soybean (Glycine max) 8 Day Old Root src2c.pk001.k1 InfectedWith Cyst Nematode sls1c Soybean (Glycine max) Infected Withsls1c.pk018.k17 Sclerotinia sclerotiorum Mycelium sah1c Soybean (Glycinemax) Sprayed With sah1c.pk002.f8 Authority ™ Herbicide

[0091] cDNA libraries may be prepared by any one of many methodsavailable. For example, the cDNAs may be introduced into plasmid vectorsby first preparing the CDNA libraries in Uni-ZAP™ XR vectors accordingto the manufacturer's protocol (Stratagene Cloning Systems, La Jolla,Calif.). The Uni-ZAP™ XR libraries are converted into plasmid librariesaccording to the protocol provided by Stratagene. Upon conversion, cDNAinserts will be contained in the plasmid vector pBluescript. Inaddition, the cDNAs may be introduced directly into precut Bluescript IISK(+) vectors (Stratagene) using T4 DNA ligase (New England Biolabs),followed by transfection into DH10B cells according to themanufacturer's protocol (GIBCO BRL Products). Once the cDNA inserts arein plasmid vectors, plasmid DNAs are prepared from randomly pickedbacterial colonies containing recombinant pBluescript plasmids, or theinsert cDNA sequences are amplified via polymerase chain reaction usingprimers specific for vector sequences flanking the inserted cDNAsequences. Amplified insert DNAs or plasmid DNAs are sequenced indye-primer sequencing reactions to generate partial cDNA sequences(expressed sequence tags or “ESTs”; see Adams et al., (1991) Science252:1651-1656). The resulting ESTs are analyzed using a Perkin ElmerModel 377 fluorescent sequencer.

[0092] Full-insert sequence (FIS) data is generated utilizing a modifiedtransposition protocol. Clones identified for FIS are recovered fromarchived glycerol stocks as single colonies, and plasmid DNAs areisolated via alkaline lysis. Isolated DNA templates are reacted withvector primed M13 forward and reverse oligonucleotides in a PCR-basedsequencing reaction and loaded onto automated sequencers. Confirmationof clone identification is performed by sequence alignment to theoriginal EST sequence from which the FIS request is made.

[0093] Confirmed templates are transposed via the Primer Islandtransposition kit (PE Applied Biosystems, Foster City, Calif.) which isbased upon the Saccharomyces cerevisiae Ty1 transposable element (Devineand Boeke (1994) Nucleic Acids Res. 22:3765-3772). The in vitrotransposition system places unique binding sites randomly throughout apopulation of large DNA molecules. The transposed DNA is then used totransform DH10B electro-competent cells (Gibco BRL/Life Technologies,Rockville, Md.) via electroporation. The transposable element containsan additional selectable marker (named DHFR; Fling and Richards (1983)Nucleic Acids Res. 11:5147-5158), allowing for dual selection on agarplates of only those subclones containing the integrated transposon.Multiple subclones are randomly selected from each transpositionreaction, plasmid DNAs are prepared via alkaline lysis, and templatesare sequenced (ABI Prism dye-terminator ReadyReaction mix) outward fromthe transposition event site, utilizing unique primers specific to thebinding sites within the transposon.

[0094] Sequence data is collected (ABI Prism Collections) and assembledusing Phred/Phrap (P. Green, University of Washington, Seattle).Phred/Phrap is a public domain software program which re-reads the ABIsequence data, re-calls the bases, assigns quality values, and writesthe base calls and quality values into editable output files. The Phrapsequence assembly program uses these quality values to increase theaccuracy of the assembled sequence contigs. Assemblies are viewed by theConsed sequence editor (D. Gordon, University of Washington, Seattle).

[0095] In some of the clones the cDNA fragment corresponds to a portionof the 3′ -terminus of the gene and does not cover the entire openreading frame. In order to obtain the upstream information one of twodifferent protocols are used. The first of these methods results in theproduction of a fragment of DNA containing a portion of the desired genesequence while the second method results in the production of a fragmentcontaining the entire open reading frame. Both of these methods use tworounds of PCR amplification to obtain fragments from one or morelibraries. The libraries some times are chosen based on previousknowledge that the specific gene should be found in a certain tissue andsome times are randomly-chosen. Reactions to obtain the same gene may beperformed on several libraries in parallel or on a pool of libraries.Library pools are normally prepared using from 3 to 5 differentlibraries and normalized to a uniform dilution. In the first round ofamplification both methods use a vector-specific (forward) primercorresponding to a portion of the vector located at the 5′ -terminus ofthe clone coupled with a gene-specific (reverse) primer. The firstmethod uses a sequence that is complementary to a portion of the alreadyknown gene sequence while the second method uses a gene-specific primercomplementary to a portion of the 3′ -untranslated region (also referredto as UTR). In the second round of amplification a nested set of primersis used for both methods. The resulting DNA fragment is ligated into apBluescript vector using a commercial kit and following themanufacturer's protocol. This kit is selected from many available fromseveral vendors including Invitrogen (Carlsbad, Calif.), Promega Biotech(Madison, Wis.), and Gibco-BRL (Gaithersburg, Md.). The plasmid DNA isisolated by alkaline lysis method and submitted for sequencing andassembly using Phred/Phrap, as above.

Example 2 Identification of cDNA Clones

[0096] cDNA clones encoding chalcone isomerase were identified byconducting BLAST (Basic Local Alignment Search Tool; Altschul et al.(1993) J. Mol. Biol. 215:403-410; see also the explanation of the BLASTalogarithm on the world wide web site for the National Center forBiotechnology Information at the National Library of Medicine of theNational Institutes of Health) searches for similarity to sequencescontained in the BLAST “nr” database (comprising all non-redundantGenBankT™ CDS translations, sequences derived from the 3-dimensionalstructure Brookhaven Protein Data Bank, the last major release of theSWISS-PROT protein sequence database, EMBL, and DDBJ databases). ThecDNA sequences obtained in Example 1 were analyzed for similarity to allpublicly available DNA sequences contained in the “nr” database usingthe BLASTN algorithm provided by the National Center for BiotechnologyInformation (NCBI). The DNA sequences were translated in all readingframes and compared for similarity to all publicly available proteinsequences contained in the “nr” database using the BLASTX algorithm(Gish and States (1993) Nat Genet. 3:266-272) provided by the NCBI. Forconvenience, the P-value (probability) of observing a match of a cDNAsequence to a sequence contained in the searched databases merely bychance as calculated by BLAST are reported herein as “pLog” values,which represent the negative of the logarithm of the reported P-value.Accordingly, the greater the pLog value, the greater the likelihood thatthe cDNA sequence and the BLAST “hit” represent homologous proteins.

[0097] ESTs submitted for analysis are compared to the GenBank™ databaseas described above. ESTs that contain sequences more 5- or 3-prime canbe found by using the BLASTn algorithm (Altschul et al (1997) NucleicAcids Res. 25:3389-3402) against the Du Pont proprietary databasecomparing nucleotide sequences that share common or overlapping regionsof sequence homology. Where common or overlapping sequences existbetween two or more nucleic acid fragments, the sequences can beassembled into a single contiguous nucleotide sequence, thus extendingthe original fragment in either the 5 or 3 prime direction. Once themost 5-prime EST is identified, its complete sequence can be determinedby Full Insert Sequencing as described in Example 1. Homologous genesbelonging to different species can be found by comparing the amino acidsequence of a known gene (from either a proprietary source or a publicdatabase) against an EST database using the tBLASTn algorithm. ThetBLASTn algorithm searches an amino acid query against a nucleotidedatabase that is translated in all 6 reading frames. This search allowsfor differences in nucleotide codon usage between different species, andfor codon degeneracy.

Example 3 Characterization of cDNA Clones Encoding Chalcone Isomerase

[0098] The BLASTX search using the sequences from clones listed in Table3 revealed similarity of the polypeptides encoded by the cDNAs tochalcone isomerase from Phaseolus vulgaris (NCBI General Identifier No.3041652; SEQ ID NO:7) and Vitis vinifera (NCBI General Identifier No.1705761; SEQ ID NO:8). Shown in Table 3 are the BLAST results forsequences encoding an entire protein derived from an FIS, a contig, oran FIS and PCR (“CGS”): TABLE 3 BLAST Results for Sequences EncodingPolypeptides Homologous to Chalcone Isomerase NCBI General IdentifierNo. BLAST Clone Status (SEQ ID NO:) pLog Score src2c.pk001.k1 CGS3041652 76.52 (SEQ ID NO: 7) sls1c.pk018.k17 CGS 3041652 77.40 (SEQ IDNO: 7) sah1c.pk002.f8 CGS 1705761 75.40 (SEQ ID NO: 8)

[0099] The nucleotide sequence of clone src2c.pk001. k1 is shown in SEQID NO:1. The amino acid sequence deduced from nucleotides 62 through 742of SEQ ID NO:1 is shown in SEQ ID NO:2. The nucleotide sequence of clonesls1c.pk018.k17 is shown in SEQ ID NO:3. The amino acid sequence deducedfrom nucleotides 13 through 693 of SEQ ID NO:3 is shown in SEQ ID NO:4.The nucleotide sequence of clone sah1c.pk002.f8 is shown in SEQ ID NO:5.The amino acid sequence deduced from nucleotides 1 through 687 of SEQ IDNO:5 is shown in SEQ ID NO:6.

[0100]FIG. 2 presents an alignment of the amino acid sequences set forthin SEQ ID NOs:2, 4, 6 and the sequence from Phaseolus vulgaris (NCBIGeneral Identifier No. 3041652; SEQ ID NO:7) and Vitis vinifera (NCBIGeneral Identifier No. 1705761; SEQ ID NO:8). The data in Table 4represents a calculation of the percent identity of the amino acidsequences set forth in SEQ ID NOs:2, 4, 6 and the sequence fromPhaseolus vulgaris (NCBI General Identifier No. 3041652; SEQ ID NO:7)and Vitis vinifera (NCBI General Identifier No. 1705761; SEQ ID NO:8).TABLE 4 Percent Identity of Amino Acid Sequences Deduced From theNucleotide Sequences of cDNA Clones Encoding Polypeptides Homologous toChalcone Isomerase NCBI SEQ ID General Identifier No. Percent Clone NO:(SEQ ID NO:) Identity src2c.pk001.k1 2 3041652 65.6 (SEQ ID NO: 7)sls1c.pk018.k17 4 3041652 66.5 (SEQ ID NO: 7) sah1c.pk002.f8 6 170576162.9 (SEQ ID NO: 8)

[0101] Sequence alignments and percent identity calculations wereperformed using the Megalign program of the LASERGENE bioinformaticscomputing suite (DNASTAR Inc., Madison, Wis.). Multiple alignment of thesequences was performed using the Clustal method of alignment (Higginsand Sharp (1989) CABIOS. 5:151-153) with the default parameters (GAPPENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwisealignments using the Clustal method were KTUPLE 1, GAP PENALTY=3,WINDOW=5 and DIAGONALS SAVED=5. Sequence alignments and BLAST scores andprobabilities indicate that the nucleic acid fragments comprising theinstant cDNA clones encode complete amino acid sequences of a chalconeisomerase.

Example 4 Expression of Recombinant DNA Constructs in Monocot Cells

[0102] A recombinant DNA construct comprising a cDNA encoding theinstant polypeptides in sense orientation with respect to the maize 27kD zein promoter that is located 5′ to the cDNA fragment, and the 10 kDzein 3′ end that is located 3′ to the cDNA fragment, can be constructed.The cDNA fragment of this gene may be generated by polymerase chainreaction (PCR) of the cDNA clone using appropriate oligonucleotideprimers. Cloning sites (Ncol or Smal) can be incorporated into theoligonucleotides to provide proper orientation of the DNA fragment wheninserted into the digested vector pML103 as described below.Amplification is then performed in a standard PCR. The amplified DNA isthen digested with restriction enzymes Ncol and Smal and fractionated onan agarose gel. The appropriate band can be isolated from the gel andcombined with a 4.9 kb Ncol-Smal fragment of the plasmid pML103. PlasmidpML103 has been deposited under the terms of the Budapest Treaty at ATCC(American Type Culture Collection, 10801 University Blvd., Manassas, Va.20110-2209), and bears accession number ATCC 97366. The DNA segment frompML103 contains a 1.05 kb Sall-Ncol promoter fragment of the maize 27 kDzein gene and a 0.96 kb Smal-Sall fragment from the 3′ end of the maize10 kD zein gene in the vector pGem9Zf(+) (Promega). Vector and insertDNA can be ligated at 15° C. overnight, essentially as described(Maniatis). The ligated DNA may then be used to transform E. coliXL1-Blue (Epicurian Coli XL-1 Blue™; Stratagene). Bacterialtransformants can be screened by restriction enzyme digestion of plasmidDNA and limited nucleotide sequence analysis using the dideoxy chaintermination method (Sequenase™ DNA Sequencing Kit; U.S. Biochemical).The resulting plasmid construct would comprise a recombinant DNAconstruct encoding, in the 5′ to 3′ direction, the maize 27 kD zeinpromoter, a cDNA fragment encoding the instant polypeptides, and the 10kD zein 3′ region.

[0103] The recombinant DNA construct described above can then beintroduced into corn cells by the following procedure. Immature cornembryos can be dissected from developing caryopses derived from crossesof the inbred corn lines H99 and LH132. The embryos are isolated 10 to11 days after pollination when they are 1.0 to 1.5 mm long. The embryosare then placed with the axis-side facing down and in contact withagarose-solidified N6 medium (Chu et al. (1975) Sci. Sin. Peking18:659-668). The embryos are kept in the dark at 27° C. Friableembryogenic callus consisting of undifferentiated masses of cells withsomatic proembryoids and embryoids borne on suspensor structuresproliferates from the scutellum of these immature embryos. Theembryogenic callus isolated from the primary explant can be cultured onN6 medium and sub-cultured on this medium every 2 to 3 weeks.

[0104] The plasmid, p35S/Ac (obtained from Dr. Peter Eckes, Hoechst Ag,Frankfurt, Germany) may be used in transformation experiments in orderto provide for a selectable marker. This plasmid contains the Pat gene(see European Patent Publication 0 242 236) which encodesphosphinothricin acetyl transferase (PAT). The enzyme PAT confersresistance to herbicidal glutamine synthetase inhibitors such asphosphinothricin. The pat gene in p35S/Ac is under the control of the35S promoter from cauliflower mosaic virus (Odell et al. (1985) Nature313:810-812) and the 3′ region of the nopaline synthase gene from theT-DNA of the Ti plasmid of Agrobacterium tumefaciens.

[0105] The particle bombardment method (Klein et al. (1987) Nature327:70-73) may be used to transfer genes to the callus culture cells.According to this method, gold particles (1 μm in diameter) are coatedwith DNA using the following technique. Ten μg of plasmid DNAs are addedto 50 μL of a suspension of gold particles (60 mg per mL). Calciumchloride (50 μL of a 2.5 M solution) and spermidine free base (20 μL ofa 1.0 M solution) are added to the particles. The suspension is vortexedduring the addition of these solutions. After 10 minutes, the tubes arebriefly centrifuged (5 sec at 15,000 rpm) and the supernatant removed.The particles are resuspended in 200 μL of absolute ethanol, centrifugedagain and the supernatant removed. The ethanol rinse is performed againand the particles resuspended in a final volume of 30 μL of ethanol. Analiquot (5 μL) of the DNA-coated gold particles can be placed in thecenter of a Kapton™ flying disc (Bio-Rad Labs). The particles are thenaccelerated into the corn tissue with a BiolisticTM PDS-1000/He (Bio-RadInstruments, Hercules Calif.), using a helium pressure of 1000 psi, agap distance of 0.5 cm and a flying distance of 1.0 cm.

[0106] For bombardment, the embryogenic tissue is placed on filter paperover agarose-solidified N6 medium. The tissue is arranged as a thin lawnand covered a circular area of about 5 cm in diameter. The petri dishcontaining the tissue can be placed in the chamber of the PDS-1000/Heapproximately 8 cm from the stopping screen. The air in the chamber isthen evacuated to a vacuum of 28 inches of Hg. The macrocarrier isaccelerated with a helium shock wave using a rupture membrane thatbursts when the He pressure in the shock tube reaches 1000 psi.

[0107] Seven days after bombardment the tissue can be transferred to N6medium that contains bialophos (5 mg per liter) and lacks casein orproline. The tissue continues to grow slowly on this medium. After anadditional 2 weeks the tissue can be transferred to fresh N6 mediumcontaining bialophos. After 6 weeks, areas of about 1 cm in diameter ofactively growing callus can be identified on some of the platescontaining the bialophos-supplemented medium. These calli may continueto grow when sub-cultured on the selective medium.

[0108] Plants can be regenerated from the transgenic callus by firsttransferring clusters of tissue to N6 medium supplemented with 0.2 mgper liter of 2,4-D. After two weeks the tissue can be transferred toregeneration medium (Fromm et al. (1990) Bio/Technology 8:833-839).

Example 5 Expression of Recombinant DNA Constructs in Dicot Cells

[0109] A seed-specific expression cassette composed of the promoter andtranscription terminator from the gene encoding the β subunit of theseed storage protein phaseolin from the bean Phaseolus vulgaris (Doyleet al. (1986) J. Biol. Chem. 261:9228-9238) can be used for expressionof the instant polypeptides in transformed soybean. The phaseolincassette includes about 500 nucleotides upstream (5′) from thetranslation initiation codon and about 1650 nucleotides downstream (3′)from the translation stop codon of phaseolin. Between the 5′ and 3′regions are the unique restriction endonuclease sites Ncol (whichincludes the ATG translation initiation codon), Smal, Kpnl and Xbal. Theentire cassette is flanked by HindIII sites.

[0110] The cDNA fragment of this gene may be generated by polymerasechain reaction (PCR) of the cDNA clone using appropriate oligonucleotideprimers. Cloning sites can be incorporated into the oligonucleotides toprovide proper orientation of the DNA fragment when inserted into theexpression vector. Amplification is then performed as described above,and the isolated fragment is inserted into a pUC18 vector carrying theseed expression cassette.

[0111] Soybean embryos may then be transformed with the expressionvector comprising sequences encoding the instant polypeptides. To inducesomatic embryos, cotyledons, 3-5 mm in length dissected from surfacesterilized, immature seeds of the soybean cultivar A2872, can becultured in the light or dark at 26° C. on an appropriate agar mediumfor 6-10 weeks. Somatic embryos which produce secondary embryos are thenexcised and placed into a suitable liquid medium. After repeatedselection for clusters of somatic embryos which multiplied as early,globular staged embryos, the suspensions are maintained as describedbelow.

[0112] Soybean embryogenic suspension cultures can be maintained in 35mL liquid media on a rotary shaker, 150 rpm, at 26° C. with florescentlights on a 16:8 hour day/night schedule. Cultures are subcultured everytwo weeks by inoculating approximately 35 mg of tissue into 35 mL ofliquid medium.

[0113] Soybean embryogenic suspension cultures may then be transformedby the method of particle gun bombardment (Klein et al. (1987) Nature(London) 327:70-73, U.S. Pat. No. 4,945,050). A DuPont Biolistic™PDS1000/HE instrument (helium retrofit) can be used for thesetransformations.

[0114] A selectable marker gene which can be used to facilitate soybeantransformation is a chimeric gene composed of the 35S promoter fromcauliflower mosaic virus (Odell et al. (1985) Nature 313:810-812), thehygromycin phosphotransferase gene from plasmid pJR225 (from E. coli;Gritz et al. (1983) Gene 25:179-188) and the 3′ region of the nopalinesynthase gene from the T-DNA of the Ti plasmid of Agrobacteriumtumefaciens. The seed expression cassette comprising the phaseolin 5′region, the fragment encoding the instant polypeptides and the phaseolin3′ region can be isolated as a restriction fragment. This fragment canthen be inserted into a unique restriction site of the vector carryingthe marker gene.

[0115] To 50 μL of a 60 mg/mL 1 μm gold particle suspension is added (inorder): 5 μL DNA (1 μg/μL), 20 μL spermidine (0.1 M), and 50 μL CaCl₂(2.5 M). The particle preparation is then agitated for three minutes,spun in a microfuge for 10 seconds and the supernatant removed. TheDNA-coated particles are then washed once in 400 μL 70% ethanol andresuspended in 40 μL of anhydrous ethanol. The DNA/particle suspensioncan be sonicated three times for one second each. Five μL of theDNA-coated gold particles are then loaded on each macro carrier disk.

[0116] Approximately 300-400 mg of a two-week-old suspension culture isplaced in an empty 60×15 mm petri dish and the residual liquid removedfrom the tissue with a pipette. For each transformation experiment,approximately 5-10 plates of tissue are normally bombarded. Membranerupture pressure is set at 1100 psi and the chamber is evacuated to avacuum of 28 inches mercury. The tissue is placed approximately 3.5inches away from the retaining screen and bombarded three times.Following bombardment, the tissue can be divided in half and placed backinto liquid and cultured as described above.

[0117] Five to seven days post bombardment, the liquid media may beexchanged with fresh media, and eleven to twelve days post bombardmentwith fresh media containing 50 mg/mL hygromycin. This selective mediacan be refreshed weekly. Seven to eight weeks post bombardment, green,transformed tissue may be observed growing from untransformed, necroticembryogenic clusters. Isolated green tissue is removed and inoculatedinto individual flasks to generate new, clonally propagated, transformedembryogenic suspension cultures. Each new line may be treated as anindependent transformation event. These suspensions can then besubcultured and maintained as clusters of immature embryos orregenerated into whole plants by maturation and germination ofindividual somatic embryos.

Example 6 Expression of Recombinant DNA Constructs in Microbial Cells

[0118] The cDNAs encoding the instant polypeptides can be inserted intothe T7 E. coli expression vector pBT430. This vector is a derivative ofpET-3a (Rosenberg et al. (1987) Gene56:125-135) which employs thebacteriophage T7 RNA polymerase/T7 promoter system. Plasmid pBT430 wasconstructed by first destroying the EcoRI and HindIII sites in pET-3a attheir original positions. An oligonucleotide adaptor containing EcoRIand Hind IlI sites was inserted at the BamHI site of pET-3a. Thiscreated pET-3aM with additional unique cloning sites for insertion ofgenes into the expression vector. Then, the Ndel site at the position oftranslation initiation was converted to an Ncol site usingoligonucleotide-directed mutagenesis. The DNA sequence of pET-3aM inthis region, 5′-CATATGG, was converted to 5′-CCCATGG in pBT430.

[0119] Plasmid DNA containing a cDNA may be appropriately digested torelease a nucleic acid fragment encoding the protein. This fragment maythen be purified on a 1% low melting agarose gel. Buffer and agarosecontain 10 μg/ml ethidium bromide for visualization of the DNA fragment.The fragment can then be purified from the agarose gel by digestion withGELase™ (Epicentre Technologies, Madison, Wis.) according to themanufacturer's instructions, ethanol precipitated, dried and resuspendedin 20 μL of water. Appropriate oligonucleotide adapters may be ligatedto the fragment using T4 DNA ligase (New England Biolabs (NEB), Beverly,Mass.). The fragment containing the ligated adapters can be purifiedfrom the excess adapters using low melting agarose as described above.The vector pBT430 is digested, dephosphorylated with alkalinephosphatase (NEB) and deproteinized with phenol/chloroform as describedabove. The prepared vector pBT430 and fragment can then be ligated at16° C. for 15 hours followed by transformation into DH5 electrocompetentcells (GIBCO BRL). Transformants can be selected on agar platescontaining LB media and 100 μg/mL ampicillin. Transformants containingthe gene encoding the instant polypeptides are then screened for thecorrect orientation with respect to the T7 promoter by restrictionenzyme analysis.

[0120] For high level expression, a plasmid clone with the cDNA insertin the correct orientation relative to the T7 promoter can betransformed into E. coli strain BL21 (DE3) (Studier et al. (1986) J.Mol. Biol. 189:113-130). Cultures are grown in LB medium containingampicillin (100 mg/L) at 25° C. At an optical density at 600 nm ofapproximately 1, IPTG (isopropylthio-β-galactoside, the inducer) can beadded to a final concentration of 0.4 mM and incubation can be continuedfor 3 h at 25° C. Cells are then harvested by centrifugation andre-suspended in 50 μL of 50 mM Tris-HCI at pH 8.0 containing 0.1 mM DTTand 0.2 mM phenyl methylsulfonyl fluoride. A small amount of 1 mm glassbeads can be added and the mixture sonicated 3 times for about 5 secondseach time with a microprobe sonicator. The mixture is centrifuged andthe protein concentration of the supernatant determined. One μg ofprotein from the soluble fraction of the culture can be separated bySDS-polyacrylamide gel electrophoresis. Gels can be observed for proteinbands migrating at the expected molecular weight.

[0121] For example, assays for chalcone isomerase are presented by Jezet al., (Nature Struct Biol. 7(9):786-791 (2000)). In addition, thereare other assays for the detection of chalcone isomerase activity (Molet al., Phytochemistry 24(10), 2267-2269 (1985); Britsch et al.,Phytochemistry24(9), 1975-6 (1985)).

1 8 1 844 DNA Glycine max 1 gtctccattt gactacaaaa cagtctccat ttgacacagtcctagtggtg tactacttat 60 tatggccaca ccagcatcca tcactaatgt cactgtggagttccttcaat tccccgcact 120 ggtgacacct cctggctcta ccaaatccta tttccttggcggcgcagggg tgagagggtt 180 gaatattcaa gaagaatttg tgaagttcac gggaataggtgtttatttgg aagacaaggc 240 cgtgtcatca ctcgctgcca aatggaaggg caagagtgcagctgaattgc tcgactccct 300 tgacttctac agagatatca tcaaaggccc ctttgagaagttaattcgag ggtcaaagtt 360 aagaacattg gatggtcgtg aatacgtaag gaaggtatcagagaactgtg tggcacatat 420 gcaatctgtt gggacttaca gtgatgagga ggaaaaagctattgaggaat ttagaaatgc 480 tttcaaggat caaaatttcc caccaggctc cactgttttctacaaacaat cacccactgg 540 aacattgggg cttagtttct cgaaagatga gacaataccagaacatgagc atgcagtgat 600 agacaacaag ccactttcgg aggcagtgct ggagactatgatcggagaga ttcctgtttc 660 ccctgctttg aaagagagtt tggctacaag gtttcatcagttcttcaaag agttagaggc 720 caatcccaac attgaaaact gagagttgca gcagttgccaactatcttta gaagaccttg 780 gaaacaagaa gaagaacagc aaaaatttga agagacatctaaataatgta cccccccccc 840 cccc 844 2 226 PRT Glycine max 2 Met Ala ThrPro Ala Ser Ile Thr Asn Val Thr Val Glu Phe Leu Gln 1 5 10 15 Phe ProAla Leu Val Thr Pro Pro Gly Ser Thr Lys Ser Tyr Phe Leu 20 25 30 Gly GlyAla Gly Val Arg Gly Leu Asn Ile Gln Glu Glu Phe Val Lys 35 40 45 Phe ThrGly Ile Gly Val Tyr Leu Glu Asp Lys Ala Val Ser Ser Leu 50 55 60 Ala AlaLys Trp Lys Gly Lys Ser Ala Ala Glu Leu Leu Asp Ser Leu 65 70 75 80 AspPhe Tyr Arg Asp Ile Ile Lys Gly Pro Phe Glu Lys Leu Ile Arg 85 90 95 GlySer Lys Leu Arg Thr Leu Asp Gly Arg Glu Tyr Val Arg Lys Val 100 105 110Ser Glu Asn Cys Val Ala His Met Gln Ser Val Gly Thr Tyr Ser Asp 115 120125 Glu Glu Glu Lys Ala Ile Glu Glu Phe Arg Asn Ala Phe Lys Asp Gln 130135 140 Asn Phe Pro Pro Gly Ser Thr Val Phe Tyr Lys Gln Ser Pro Thr Gly145 150 155 160 Thr Leu Gly Leu Ser Phe Ser Lys Asp Glu Thr Ile Pro GluHis Glu 165 170 175 His Ala Val Ile Asp Asn Lys Pro Leu Ser Glu Ala ValLeu Glu Thr 180 185 190 Met Ile Gly Glu Ile Pro Val Ser Pro Ala Leu LysGlu Ser Leu Ala 195 200 205 Thr Arg Phe His Gln Phe Phe Lys Glu Leu GluAla Asn Pro Asn Ile 210 215 220 Glu Asn 225 3 815 DNA Glycine max 3atttgacaca ttatggccac accagcatcc atcaccaatg tcactgtgga gttccttcaa 60ttccccgcgg tggtgacacc tcctgcctct accaaatcct atttccttgg tggcgcaggg 120gtgagagggt tgaatattga agaagaattt gtaaagttca cgggaatagg tgtttatttg 180gaagacaagg ccgtgtcatc actcggtgcc aaatggaagg gcaaaagtgc agctgaattg 240ctggactcac ttgacttcta cagagatatc atcaaaggtc cgtttgagaa gttaattcga 300gggtcaaagt taagaacatt ggatggtcgt gaatacgtaa ggaaggtgtc agagaactgt 360gtggcccata tggaatccgt tgggacttac agtgaagcag aagaaaaagc tattgaggaa 420tttagaaatg ctttcaagga tcaaaatttc ccaccaggct ccactgtttt ctacaaacaa 480tcacccactg gaacattggg gcttagtttc tcgaaagatg agacaatacc agaacatgag 540catgcagtga tagacaacaa accactctcg gaggccgttc tggagactat gatcggagag 600attcctgttt cccctgcttt aaaagagagt ttggctacaa ggtttcacca gtttttcaaa 660gagttagagg ccaatcccaa caatgaaaac tgagaacaat ctttagggga cattggaaac 720aggaacaaca aaaaaatccc cactttgttt gaatttgtac cccatttaat aaatgagcaa 780accagttaga taataaaatt tataaattag aaaat 815 4 226 PRT Glycine max 4 MetAla Thr Pro Ala Ser Ile Thr Asn Val Thr Val Glu Phe Leu Gln 1 5 10 15Phe Pro Ala Val Val Thr Pro Pro Ala Ser Thr Lys Ser Tyr Phe Leu 20 25 30Gly Gly Ala Gly Val Arg Gly Leu Asn Ile Glu Glu Glu Phe Val Lys 35 40 45Phe Thr Gly Ile Gly Val Tyr Leu Glu Asp Lys Ala Val Ser Ser Leu 50 55 60Gly Ala Lys Trp Lys Gly Lys Ser Ala Ala Glu Leu Leu Asp Ser Leu 65 70 7580 Asp Phe Tyr Arg Asp Ile Ile Lys Gly Pro Phe Glu Lys Leu Ile Arg 85 9095 Gly Ser Lys Leu Arg Thr Leu Asp Gly Arg Glu Tyr Val Arg Lys Val 100105 110 Ser Glu Asn Cys Val Ala His Met Glu Ser Val Gly Thr Tyr Ser Glu115 120 125 Ala Glu Glu Lys Ala Ile Glu Glu Phe Arg Asn Ala Phe Lys AspGln 130 135 140 Asn Phe Pro Pro Gly Ser Thr Val Phe Tyr Lys Gln Ser ProThr Gly 145 150 155 160 Thr Leu Gly Leu Ser Phe Ser Lys Asp Glu Thr IlePro Glu His Glu 165 170 175 His Ala Val Ile Asp Asn Lys Pro Leu Ser GluAla Val Leu Glu Thr 180 185 190 Met Ile Gly Glu Ile Pro Val Ser Pro AlaLeu Lys Glu Ser Leu Ala 195 200 205 Thr Arg Phe His Gln Phe Phe Lys GluLeu Glu Ala Asn Pro Asn Asn 210 215 220 Glu Asn 225 5 787 DNA Glycinemax 5 atggcaatgg catttccgtc cgtaacctct gtaactgtgg agaacgtgac attccctccc60 accgtgaagc ctccctgctc ccccaacacc ttcttcctcg ccggcgccgg cgtcaggggc 120ctccaaattc atcacgcttt cgttaaattc actgccatct gcatttacct ccaatacgac 180gccctttcct tcctctccgt taagtggaaa accaagtcta ctcaccagtt aacggaatcc 240gaccaattct tctcagatat cgttacaggt ccattcgaga aatttatgca ggtgacaatg 300atcaaaccct tgacggggca gcaatactca gagaaagtgg cagaaaattg tgtggccatt 360tggaggtctc ttgggattta tacagattca gaagccgaag caatagacaa gtttctttct 420gttttcaaag acctaacatt cccaccaggc tcctctatcc ttttcactgt ctcacccaat 480ggatcattaa cgataagttt ctctggtgat gaaaccattc cagaagttac gagtgctgtt 540atagagaata aactactctc agaggctgtg ttggagtcaa tgataggtaa gaatggcgtt 600tcccctgcag caaaacagag tttggcctcc agattatctc acttattcaa agagcctggg 660gtttgtgacc ctcaatctca taagtgaact cattatatca tcaacccaag atcaaaatcc 720cttaccactc tgctgtgact ttcagtactg tgctactaat aaataaaggg caaattacag 780tttttct 787 6 228 PRT Glycine max 6 Met Ala Met Ala Phe Pro Ser Val ThrSer Val Thr Val Glu Asn Val 1 5 10 15 Thr Phe Pro Pro Thr Val Lys ProPro Cys Ser Pro Asn Thr Phe Phe 20 25 30 Leu Ala Gly Ala Gly Val Arg GlyLeu Gln Ile His His Ala Phe Val 35 40 45 Lys Phe Thr Ala Ile Cys Ile TyrLeu Gln Tyr Asp Ala Leu Ser Phe 50 55 60 Leu Ser Val Lys Trp Lys Thr LysSer Thr His Gln Leu Thr Glu Ser 65 70 75 80 Asp Gln Phe Phe Ser Asp IleVal Thr Gly Pro Phe Glu Lys Phe Met 85 90 95 Gln Val Thr Met Ile Lys ProLeu Thr Gly Gln Gln Tyr Ser Glu Lys 100 105 110 Val Ala Glu Asn Cys ValAla Ile Trp Arg Ser Leu Gly Ile Tyr Thr 115 120 125 Asp Ser Glu Ala GluAla Ile Asp Lys Phe Leu Ser Val Phe Lys Asp 130 135 140 Leu Thr Phe ProPro Gly Ser Ser Ile Leu Phe Thr Val Ser Pro Asn 145 150 155 160 Gly SerLeu Thr Ile Ser Phe Ser Gly Asp Glu Thr Ile Pro Glu Val 165 170 175 ThrSer Ala Val Ile Glu Asn Lys Leu Leu Ser Glu Ala Val Leu Glu 180 185 190Ser Met Ile Gly Lys Asn Gly Val Ser Pro Ala Ala Lys Gln Ser Leu 195 200205 Ala Ser Arg Leu Ser His Leu Phe Lys Glu Pro Gly Val Cys Asp Pro 210215 220 Gln Ser His Lys 225 7 221 PRT Phaseolus vulgaris 7 Met Ala ThrAla Pro Thr Ile Thr Asp Val Gln Val Glu Phe Leu His 1 5 10 15 Phe ProAla Val Val Thr Ser Pro Ala Thr Ala Lys Thr Tyr Phe Leu 20 25 30 Gly GlyAla Gly Glu Arg Gly Leu Thr Ile Glu Gly Lys Phe Ile Lys 35 40 45 Phe ThrAla Ile Gly Val Tyr Leu Glu Asp Lys Ala Val Ala Ser Leu 50 55 60 Ala ThrLys Trp Lys Gly Lys Pro Ser Glu Glu Leu Ile Asn Thr Leu 65 70 75 80 AspPhe Tyr Arg Asp Ile Ile Ser Gly Pro Phe Glu Lys Leu Ile Arg 85 90 95 GlySer Lys Ile Leu Gln Leu Ser Gly Thr Glu Tyr Ser Arg Lys Val 100 105 110Met Glu Asn Cys Val Ala His Leu Lys Ser Val Gly Thr Tyr Gly Asp 115 120125 Ala Glu Ala Lys Gly Ile Glu Glu Phe Ala Glu Ala Phe Lys Lys Val 130135 140 Asn Phe Pro Pro Gly Ala Ser Val Phe Tyr Arg Gln Ser Pro Asp Gly145 150 155 160 Ile Leu Gly Leu Ser Phe Ser Glu Asp Ala Thr Ile Pro GlyGlu Glu 165 170 175 Ala Val Val Ile Glu Asn Lys Ala Val Ser Ala Ala ValLeu Glu Thr 180 185 190 Met Ile Gly Glu His Ala Val Ser Pro Asp Leu LysArg Ser Leu Ala 195 200 205 Ser Arg Leu Pro Ala Val Leu Asn Gly Gly IleIle Val 210 215 220 8 234 PRT Vitis vinifera 8 Met Ser Gln Val Pro SerVal Thr Ala Val Gln Val Glu Asn Val Leu 1 5 10 15 Phe Pro Pro Ser ValLys Pro Pro Gly Ser Thr Asn Asp Leu Phe Leu 20 25 30 Gly Gly Ala Gly ValArg Gly Leu Glu Ile Gln Gly Lys Phe Val Lys 35 40 45 Phe Thr Ala Ile GlyVal Tyr Leu Glu Asn Ser Ala Val Pro Thr Leu 50 55 60 Ala Val Lys Trp LysGly Lys Thr Val Glu Glu Leu Ala Asp Ser Val 65 70 75 80 Asp Phe Phe ArgAsp Val Val Thr Gly Pro Phe Glu Lys Phe Thr Lys 85 90 95 Val Thr Thr IleLeu Pro Leu Thr Gly Arg Gln Tyr Ser Asp Lys Val 100 105 110 Ser Glu AsnCys Val Ala Phe Trp Lys Ser Val Gly Ile Tyr Thr Asp 115 120 125 Ala GluAla Lys Ala Ile Glu Lys Phe Asn Glu Val Leu Lys Asp Glu 130 135 140 ThrPhe Pro Pro Gly Asn Ser Ile Leu Phe Thr His Ser Pro Leu Gly 145 150 155160 Ala Leu Thr Met Ser Phe Ser Lys Asp Gly Ser Leu Pro Glu Val Gly 165170 175 Asn Ala Val Ile Glu Asn Lys Leu Leu Thr Glu Ala Val Leu Glu Ser180 185 190 Ile Ile Gly Lys His Gly Val Ser Pro Glu Ala Lys Lys Ser LeuAla 195 200 205 Ala Arg Leu Ser Glu Leu Phe Cys Lys Glu Ala Gly Asp GluLys Ile 210 215 220 Glu Ala Glu Lys Val Ala Pro Val Ala Cys 225 230

What is claimed is:
 1. An isolated polynucleotide comprising: (a) anucleotide sequence encoding a polypeptide having chalcone isomeraseactivity, wherein the amino acid sequence of the polypeptide and theamino acid sequence of SEQ ID NO:2, 4 or 6 have at least 80% sequenceidentity, or (b) the full-length complement of the nucleotide sequenceof (a).
 2. The polynucleotide of claim 1, wherein the amino acidsequence of the polypeptide and the amino acid sequence of SEQ ID NO:2,4 or 6 have at least 85% identity.
 3. The polynucleotide of claim 1,wherein the amino acid sequence of the polypeptide and the amino acidsequence of SEQ ID NO:2, 4 or 6 have at least 90% identity.
 4. Thepolynucleotide of claim 1, wherein the amino acid sequence of thepolypeptide and the amino acid sequence of SEQ ID NO:2, 4 or 6 have atleast 95% identity.
 5. The polynucleotide of claim 1, wherein the aminoacid sequence of the polypeptide comprises the amino acid sequence ofSEQ ID NO:2, 4 or
 6. 6. The polynucleotide of claim 1 wherein thenucleotide sequence comprises the nucleotide sequence of SEQ ID NO:1, 3or
 5. 7. A vector comprising the polynucleotide of claim
 1. 8. Arecombinant DNA construct comprising the polynucleotide of claim 1operably linked to at least one regulatory sequence.
 9. A method fortransforming a cell, comprising transforming a cell with thepolynucleotide of claim
 1. 10. A cell comprising the recombinant DNAconstruct of claim
 8. 11. A method for producing a plant comprisingtransforming a plant cell with the polynucleotide of claim 1 andregenerating a plant from the transformed plant cell.
 12. A plantcomprising the recombinant DNA construct of claim
 8. 13. A seedcomprising the recombinant DNA construct of claim
 8. 14. An isolatedpolynucleotide comprising a first nucleotide sequence, wherein the firstnucleotide sequence contains at least 30 nucleotides, and wherein thefirst nucleotide sequence is comprised by another polynucleotide,wherein the other polynucleotide includes: (a) a second nucleotidesequence, wherein the second nucleotide sequence encodes a polypeptidehaving chalcone isomerase activity, wherein the amino acid sequence ofthe polypeptide and the amino acid sequence of SEQ ID NO:2, 4 or 6having at least 80% sequence identity, or (b) the complement of thesecond nucleotide sequence of (a).
 15. An isolated polypeptide havingchalcone isomerase activity, wherein the amino acid sequence of thepolypeptide and the amino acid sequence of SEQ ID NO:2, 4 or 6 have atleast 80% identity.
 16. The polypeptide of claim 15, wherein the aminoacid sequence of the polypeptide and the amino acid sequence of SEQ IDNO:2, 4 or 6 have at least 85% identity.
 17. The polypeptide of claim15, wherein the amino acid sequence of the polypeptide and the aminoacid sequence of SEQ ID NO:2, 4 or 6 have at least 90% identity.
 18. Thepolypeptide of claim 15, wherein the amino acid sequence of thepolypeptide and the amino acid sequence of SEQ ID NO:2, 4 or 6 have atleast 95% identity.
 19. The polypeptide of claim 15, wherein the aminoacid sequence of the polypeptide comprises the amino acid sequence ofSEQ ID NO:2, 4 or
 6. 20. A method for isolating a polypeptide encoded bythe polynucleotide of claim 1 comprising isolating the polypeptide froma cell or culture medium of the cell, wherein the cell comprises arecombinant DNA construct comprising the polynucleotide of claim 1operably linked to at least one regulatory sequence.